The current state of the art for optical microscopy of living cells provides an array of techniques of extraordinary power. Living cells can now be studied in multiple dimensions (three spatial dimensions, time, multiple wavelengths, and multiple stage positions). A unique feature of microscopic approaches is the capability of observing transient, ephemeral structures and interactions on a cell-by-cell basis. This includes the ability to monitor subcellular processes and to follow cell movements as well as cell-cell interactions over time. We propose to develop an instrument that will couple the power of multidimensional microscopy with that of DNA array technology. Specifically, we envision an instrument in which individual cells selected on the basis of optically detectable features at critical time points in dynamic processes can be rapidly and robotically micromanipulated into reaction chambers to permit amplified cDNA synthesis and subsequent array analysis. In this way, "snapshots" of gene expression in single cells can be related to information obtained with multidimensional microscopy. The proposed instrument will incorporate an inverted research microscope capable of widefield deconvolution microscopy as well as a robotic system for manipulation of cells and reagents. An environmental chamber will provide conditions for optimal maintenance of cells. A laser ablation system will provide for automated cell lysis. Algorithms will be developed for automatic recognition and manipulation of cells, a requirement for high throughput. The planned system is expected to process 500-1000 cells per day. We believe that the proposed instrument will represent a genuine advance in technology that will be of great benefit to cell biology and the study of cancer cells. For example, the progression to malignancy involves the gradual accumulation of genetic changes in single cells, leading to heterogeneity among malignant cells. Studies of gene expression at the single-cell level will permit an orderly dissection of this heterogeneity. In summary, we believe that an instrument which couples multidimensional microscopy with DNA array technology will be a spectacular tool that will be useful to many laboratories.